Media and methods for enhancing the survival and proliferation of stem cells

ABSTRACT

The present disclosure relates to improved supplements, culture media and methods for enhancing the survival or proliferation of mammalian stem cells. In particular, adding a lipid supplement, such as a lipid-enriched carrier (e.g. a lipid-enriched albumin), to the culture medium may enhance the survival and/or proliferation of the stem cells by at least 5% to 65% as compared to a culture medium that does not contain the lipid supplement.

RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.16/480,025, filed Jul. 23, 2019, which is a national phase entryapplication of Patent Cooperation Treaty Application No.PCT/CA2018/050076, filed Jan. 23, 2018 (which designates the U.S.),which claims the benefit under 35 USC § 119(e) from U.S. ProvisionalApplication No. 62/449,413, filed on Jan. 23, 2017 (now abandoned),62/518,776, filed on Jun. 13, 2017 (now abandoned) and 62/608,875, filedon Dec. 21, 2017 (now abandoned), all of which are incorporated hereinby reference in their entirety.

BACKGROUND

Stem cell research has become a fast moving research field with a widevariety of potential applications ranging from the study of embryonicdevelopment, disease modelling, toxicology screening, and cell basedtherapies. Since the derivation of the first human embryonic stem celllines (ESCs), ESCs have received wide public attention owing to theirpotential use in regenerative medicine (Thomson et al. 1998). Theseemingly indefinite proliferative capability of ESCs coupled with theirability to differentiate into all somatic cell types makes them anattractive renewable resource of transplantable human tissue.

Furthermore, since the discovery that differentiated somatic cells couldbe reprogrammed to an ESC-like state by transfecting cells with stemcell transcription factors, the potential for personalised medicine wasrealised (Takahashi and Yamanaka 2006). Patient-specific somatic cellsmay be induced to pluripotent stem cell (iPSC) lines, and transplantedback into the patient in order to decrease the risk of immune-rejectionfollowing transplantation.

As with any fast moving research area, methods for generating iPSCs havebecome more efficient. Furthermore, with the emergence ofnon-integrating technologies, iPSCs have become more clinically relevant(Maeder and Gersbach 2016). Such advances coupled with the recentdevelopment of more accessible gene editing techniques (ZFNs, TALENs,CRISPR etc.) may pave the way to remedy disease-specific mutations priorto transplanting cells back into the patient.

Gene-editing is also a fast moving research field with new applicationsand modifications being published frequently. The unpredictable natureof cellular DNA-repair mechanisms following gene-editing, which in mostinstances causes double strand breaks at specific places in the genome,can lead to a population of target cells comprising different indels.This is a potential problem when trying to assess what effect thealtered genome has on the cells when different mutations could displaydifferent phenotypes. To resolve this issue a homogeneous populationcontaining the same alteration can be obtained by clonal derivation ofnew cell lines from a single cell.

The efficiency of deriving cell lines from single cells can be verychallenging, human pluripotent stem cells are one example where cloningefficiency can be very low.

Human pluripotent stem cells, unlike their murine counterparts, aredifficult to culture as single cells, seeding single cells at low toclonal densities can result in mass cell death shortly after plating.This has been attributed to certain bottlenecks following single cellseeding; of the cells that do survive the initial plating, many of whichdo not re-enter the cell cycle, and of those cells that do re-enter thecell cycle many of the daughter cells do not survive, and very few formlong-term proliferating colonies (Barbaric et al. 2014).

Approaches to solving the foregoing problems associated with the cultureof single mammalian stem cells have involved complex media formulationscomprising an array of small molecule inhibitors. Such mediaformulations are inadequate, on account of the cost of manufacture andtheir inefficiency. Accordingly, there remains a need for culture mediaand methods to enhance the survival and/or proliferation of mammalianstem cells in in vitro cultures.

SUMMARY

The present disclosure relates to improved supplements, culture mediaand methods for enhancing the survival or proliferation of mammalianstem cells. In particular, the inventors have shown that adding a lipidsupplement, such as a lipid-enriched carrier (e.g. a lipid-enrichedalbumin), to the culture medium may enhance the survival and/orproliferation of the stem cells by at least 5% to 65% as compared to aculture medium that does not contain the lipid supplement. Moreparticularly, adding a lipid supplement, such as a lipid-enrichedcarrier (e.g. a lipid-enriched albumin), to the culture medium mayenhance the survival and/or proliferation of the stem cells byapproximately 10% to 40%. An average cloning efficiency using thedescribed lipid supplement and/or culture medium is approximately 30%.

In one aspect of this disclosure a lipid supplement for enhancing thesurvival or proliferation of one or more mammalian stem cells isprovided. In one embodiment, the lipid supplement may comprise one ormore lipids. In another embodiment, the lipid supplement may compriseone or more lipids in the presence of a carrier. In a furtherembodiment, the lipid supplement may comprise a lipid-enriched carrier,such as a lipid-enriched albumin.

In another aspect of this disclosure a culture medium for enhancing thesurvival or proliferation of one or more mammalian stem cells isprovided. In one embodiment the culture medium may comprise a lipidsupplement. In one embodiment, the lipid supplement may comprise one ormore lipids. In another embodiment, the lipid supplement may compriseone or more lipids in the presence of a carrier. In a furtherembodiment, the lipid supplement of the culture medium for enhancing thesurvival or proliferation of one or more mammalian stem cells maycomprise a lipid-enriched carrier, such as a lipid-enriched albumin.

In one embodiment, the culture medium comprises a lipid supplement, suchas a lipid-enriched albumin, and one or more survival factors such asone or more small molecule inhibitors.

In another aspect, the culture medium may comprise an extracellularmatrix. In embodiments where the culture medium comprises anextracellular matrix, a concentration of the extracellular matrix may bebelow a gelation threshold thereof. In one embodiment, the extracellularmatrix comprises one or more monomatrix components. Such culture mediamay enhance the survival and/or proliferation (ie. enhance cloningefficiency, increase the number of recovered clones, and/or increase thesurvival rate) of the one or more stem cells by about 5% to about 65%.

In another embodiment, the culture medium comprising an extracellularmatrix, may optionally further comprise a lipid supplement.

In another embodiment of a culture medium for enhancing the survival orproliferation of one or more mammalian stem cells comprising anextracellular matrix, the culture medium may still further comprise oneor more survival factors.

In another embodiment, the present disclosure provides a lipidsupplement-free culture medium for enhancing the survival orproliferation of one or more mammalian stem cells wherein the mediacomprises an extracellular matrix.

In one embodiment, a concentration of the extracellular matrix is belowa gelation threshold thereof. In some embodiments the extracellularmatrix comprises one or more monomatrix components.

In another embodiment, the lipid supplement-free culture mediumcomprises one or more survival factors such as one or more smallmolecule inhibitors.

In another aspect, the present disclosure provides methods of enhancingthe survival or proliferation of mammalian stem cells comprisingculturing the stem cells in a culture medium comprising a lipid-enrichedalbumin.

In one embodiment, the culture media may comprise an extracellularmatrix. In another embodiment, a concentration of the extracellularmatrix may be below a gelation threshold thereof.

In one embodiment, culturing comprises culturing the one or moremammalian stem cells as a monolayer. In another embodiment, culturingcomprises culturing the one or more mammalian stem cells undernon-adherent conditions

In a further aspect, the present disclosure provides methods ofenhancing the survival or proliferation of one or more mammalian stemcells. In one embodiment, the method may comprise culturing the one ormore mammalian stem cells in the presence of a lipid supplement, asdescribed herein. In another embodiment, the method may compriseculturing the one or more mammalian stem cells in the presence of aculture medium, as described herein. In a further embodiment, the methodmay comprise culturing the stem cells in a lipid supplement-free culturemedium comprising an extracellular matrix.

In one embodiment, a concentration of the extracellular matrix may bebelow a gelation threshold thereof.

In one embodiment, culturing comprises culturing the one or moremammalian stem cells as a monolayer. In another embodiment, culturingcomprises culturing the one or more mammalian stem cells undernon-adherent conditions.

In one embodiment, the method for enhancing the survival orproliferation of one or more mammalian stem cells may comprise:

-   -   a) providing one or mammalian stem cells;    -   b) culturing the one or more mammalian stem cells in a culture        medium comprising a lipid supplement;    -   c) enhancing the survival or proliferation of the one or more        mammalian stem cells; and    -   d) yielding a 5%-65% cloning efficiency.

In another embodiment, the method for enhancing the survival orproliferation of one or more mammalian stem cells may comprise:

-   -   a) providing one or mammalian stem cells;    -   b) culturing the one or more mammalian stem cells in a culture        medium comprising an extracellular matrix below a gelation        threshold thereof, and optionally a lipid supplement;    -   c) enhancing the survival or proliferation of the one or more        mammalian stem cells; and    -   d) yielding a 5%-65% cloning efficiency.

Other features and advantages of the present invention will becomeapparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples while indicating preferred embodiments of the invention aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph showing the % cloning efficiency for various celllines cultured in different culture media.

FIG. 2 is a bar graph showing the % cloning efficiency for various celllines cultured in different culture media.

FIG. 3 is a graph showing the daily fold expansion for various WLS-1Chuman stem cell clones as compared to the clump passaged control.

FIG. 4 is a bar graph showing the % cloning efficiency of hPSC invarious culture media.

FIG. 5 shows representative images of wells from Example 4 stained withalkaline phosphatase.

FIG. 6 is a bar graph showing the % cloning efficiency of hPSC culturedunder non-adherent conditions in various culture media.

FIG. 7 is a bar graph showing the number of recovered clones of hPSCcultured under non-adherent conditions in various culture media.

FIG. 8 is a bar graph showing that lipid-loading different fatty acidsindividually or in combination onto a low-lipid BSA increases cloningefficiency.

FIG. 9 is a bar graph showing that adding lipids in the presence of acarrier increases cloning efficiency and does not have to bespecifically lipid-loaded.

FIGS. 10A and 10B are bar graphs showing that lipids do not need to belipid-loaded but can be added in the presence of a carrier to increasecloning efficiency of hPSCs in a protein rich medium (FIG. 10A) and aprotein free medium (FIG. 10B).

FIG. 11 is a bar graph showing that the cloning efficiency of mediacomprising BSA from multiple BSA suppliers can be improved by addingfree fatty acids. Supplier A lot 1&2, Supplier B lot 1 and Supplier Clot 1 BSAs already comprise of 3000-7000 ug/g of overall fatty acid.Rest of the suppliers BSAs contain less than 400 ug/g overall fattyacid.

FIG. 12 is a bar graph showing that fatty acids have different effectson cloning efficiency, some are beneficial whereas others aredetrimental.

FIG. 13 is a bar graph showing that adding detrimental fatty acids to alipid enriched BSA has a negative effect on cloning efficiency.

DETAILED DESCRIPTION

This disclosure relates to methods and media for enhancing the survivalor proliferation of mammalian stem cells.

Where used herein, “enhancing the survival or proliferation” meansincreased survival or proliferation of one or more cells when culturedin the presence of a lipid supplement or an extracellular matrix, orboth, in comparison to one or more cells not cultured in the presence ofthe lipid supplement or the extracellular matrix, or both, but otherwisecultured under the same or substantially the same conditions.

Where used herein, “lipid supplement” means a preparation of one or morelipids and/or lipid-like substances. The preparation may be provided asfree one or more lipids (or free one or more fatty acids) and/orlipid-like substances. Or, the preparation may be provided in thepresence of a carrier. Or, the preparation may be loaded on a carrier,forming a lipid-enriched carrier. By way of non-limiting example, thelipid-enriched carrier may be a lipid-enriched albumin. The preparationof one or more lipids and/or lipid-like substances may be loaded onto acarrier prior to exposing a cell culture of one or more mammalian stemcells thereto. Or, the preparation of one or more lipids and/orlipid-like substances may be combined with a carrier prior to exposing acell culture of one or more mammalian stem cells thereto. Or, thepreparation of one or more lipids and/or lipid-like substances and acarrier may be separately provided to a cell culture of one or moremammalian stem cells. In any embodiment the lipid supplement may beprovided to cell culture in a cell culture medium, such as a stem cellculture medium.

Where used herein, “carrier” means a biological or non-biological agent,substance, composition or complex that is capable of transporting someor all of the lipid supplement to a cell, whether in vitro or in vivo.More specifically, the carrier is capable of transporting one or morelipids to a cell, whether in vitro or in vivo. By way of non-limitingexamples, a carrier may be an albumin, a micelle, a liposome, anextracellular vesicle, an exosome, a cyclodextrin, a nanostructuredlipid carrier, or otherwise.

Where used herein, “mammalian stem cell” means a cell that may upon celldivision retain the ability to self-renew and to give rise to at leastone differentiated daughter cell. A mammalian stem cell includes apluripotent stem cell, such as: an embryonic stem cell (ESC); an inducedpluripotent stem cells (iPSC); and cells which have beentransdifferentiated whereby the arising cell may upon cell divisionretain the ability to self-renew and to give rise to at least onedifferentiated daughter cell. In a particular embodiment a mammalianstem cell also includes adult tissue stem cells and any progenitor cellswhether upstream or downstream thereof.

Lipid Supplement

In one aspect of this disclosure a lipid supplement for enhancing thesurvival or proliferation of one or more mammalian stem cells isprovided. The lipid supplement may comprise one or more lipids.

In some embodiments of the lipid supplement, the one or more lipids maybe selected from the group comprising: a fatty acid; a glycerolipid; aglycerophospholipid; a sphingolipid; a sterol lipid; a prenol lipid; asaccharolipid; or a polyketide.

In other embodiments of the lipid supplement, the one or more lipids mayinclude a lipid-like substance, such as a poloxamer. The poloxamer maybe Kolliphor™, Synperonics™, or Pluronics™. In certain embodiments, thelipid-like substance may be Kolliphor™ P188.

In other embodiments of the lipid supplement, the lipid supplement mayinclude non-lipid components such as vitamins or derivatives oranalogues thereof. Examples of vitamins that may be included in thelipid supplement include vitamin A, vitamin B, vitamin C, vitamin D, andvitamin E. Examples of vitamin derivatives or analogues may included-alpha tocopherol, d-alpha tocopheryl acetate, d-alpha tocopherylsuccinate, D-L-alpha-tocopherol, and LA2P.

In still other embodiments of the lipid supplement, the one or morelipids may be selected from the group comprising: a fatty acid; aglycerolipid; a glycerophospholipid; a sphingolipid; a sterol lipid; aprenol lipid; a saccharolipid; or a polyketide, and may also include alipid-like substance, such as a poloxamer. The poloxamer may beKolliphor™, Synperonics™, or Pluronics™. In certain embodiments, thelipid-like substance may be Kolliphor™ P188.

Still further, the lipid supplement may include non-lipid componentssuch as vitamins or derivatives or analogues thereof. Examples ofvitamins that may be included in the lipid supplement include vitamin A,vitamin B, vitamin C, vitamin D, and vitamin E. Examples of vitaminderivatives or analogues may include d-alpha tocopherol, d-alphatocopheryl acetate, d-alpha tocopheryl succinate, D-L-alpha-tocopherol,and LA2P.

In a preferred embodiment of the lipid supplement, the one or morelipids include at least one fatty acid. In a specific embodiment, theone or more lipids include more than one fatty acid. The at least onefatty acid may be a saturated fatty acid or an unsaturated fatty acid.Or, the more than one fatty acid may include a plurality of saturatedfatty acids, a plurality of unsaturated fatty acids, or a combination ofat least one saturated fatty acid and at least one unsaturated fattyacid.

In embodiments of the lipid supplement where the one or more lipidsinclude at least one fatty acid, the at least one fatty acid may beselected from the group of saturated fatty acids comprising propionicacid, butyric acid, valeric acid, caproic acid, enanthic acid, caprylicacid, pelargonic acid, capric acid, undecylic acid, lauric acid,tridecylic acid, myristic acid, pentadecylic acid, palmitic acid,margaric acid, stearic acid, nonadecylic acid, arachidic acid,heneicosylic acid, behenic acid, tricosylic acid, lignoceric acid,pentacosylic acid, cerotic acid, heptacosylic acid, montanic acid,nonacosylic acid, melissic acid, henatriacontylic acid, lacceroic acid,psyllic acid, geddic acid, ceroplastic acid, hexatriacontylic acid,heptatriacontanoic acid, or octatriacontanoic acid, and/or from thegroup of unsaturated fatty acids comprising α-linolenic acid,stearidonic acid, eicosapentaenoic acid, docosahexaenoic acid, linoleicacid, γ-linolenic acid, dihomo-γ-linolenic acid, arachidonic acid,docosatetraenoic acid, palmitoleic acid, vaccenic acid, paullinic acid,oleic acid, elaidic acid, gondoic acid, erucic acid, nervonic acid, ormead acid.

In embodiments of the lipid supplement where the one or more lipidsinclude more than one fatty acid, the plurality of saturated fatty acidsmay be selected from the group comprising propionic acid, butyric acid,valeric acid, caproic acid, enanthic acid, caprylic acid, pelargonicacid, capric acid, undecylic acid, lauric acid, tridecylic acid,myristic acid, pentadecylic acid, palmitic acid, margaric acid, stearicacid, nonadecylic acid, arachidic acid, heneicosylic acid, behenic acid,tricosylic acid, lignoceric acid, pentacosylic acid, cerotic acid,heptacosylic acid, montanic acid, nonacosylic acid, melissic acid,henatriacontylic acid, lacceroic acid, psyllic acid, geddic acid,ceroplastic acid, hexatriacontylic acid, heptatriacontanoic acid, oroctatriacontanoic acid; or the plurality of unsaturated fatty acids maybe selected from the group comprising α-linolenic acid, stearidonicacid, eicosapentaenoic acid, docosahexaenoic acid, linoleic acid,γ-linolenic acid, dihomo-γ-linolenic acid, arachidonic acid,docosatetraenoic acid, palmitoleic acid, vaccenic acid, paullinic acid,oleic acid, elaidic acid, gondoic acid, erucic acid, nervonic acid, ormead acid; or the combination of at least one saturated fatty acid andat least one unsaturated fatty acid may be selected from the groupcomprising propionic acid, butyric acid, valeric acid, caproic acid,enanthic acid, caprylic acid, pelargonic acid, capric acid, undecylicacid, lauric acid, tridecylic acid, myristic acid, pentadecylic acid,palmitic acid, margaric acid, stearic acid, nonadecylic acid, arachidicacid, heneicosylic acid, behenic acid, tricosylic acid, lignoceric acid,pentacosylic acid, cerotic acid, heptacosylic acid, montanic acid,nonacosylic acid, melissic acid, henatriacontylic acid, lacceroic acid,psyllic acid, geddic acid, ceroplastic acid, hexatriacontylic acid,heptatriacontanoic acid, octatriacontanoic acid, α-linolenic acid,stearidonic acid, eicosapentaenoic acid, docosahexaenoic acid, linoleicacid, γ-linolenic acid, dihomo-γ-linolenic acid, arachidonic acid,docosatetraenoic acid, palmitoleic acid, vaccenic acid, paullinic acid,oleic acid, elaidic acid, gondoic acid, erucic acid, nervonic acid, ormead acid.

In a specific embodiment of the lipid supplement, the one or more lipidsare selected from the group comprising Mead's acid, arachidic acid,palmitoleic acid, oleic acid, myristic acid, palmitic acid, myristoleicacid, linoleic acid, stearic acid, α-linolenic acid, arachidonic acid,cholesterol, DL-α-tocopheryl, Kolliphor P188.

In a more specific embodiment of the lipid supplement, the one or morelipids include three or more of palmitic acid, stearic acid, oleic acid,linoleic acid, and α-linolenic acid.

In a still more specific embodiment of the lipid supplement, the one ormore lipids of the lipid supplement include palmitic acid and oleicacid.

In embodiments where the lipid supplement may include more than onefatty acid, the more than one fatty acid may include a saturated fattyacid or an unsaturated fatty acid, or both. The more than one fatty acidmay be selected from the group comprising propionic acid, butyric acid,valeric acid, caproic acid, enanthic acid, caprylic acid, pelargonicacid, capric acid, undecylic acid, lauric acid, tridecylic acid,myristic acid, pentadecylic acid, palmitic acid, margaric acid, stearicacid, nonadecylic acid, arachidic acid, heneicosylic acid, behenic acid,tricosylic acid, lignoceric acid, pentacosylic acid, cerotic acid,heptacosylic acid, montanic acid, nonacosylic acid, melissic acid,henatriacontylic acid, lacceroic acid, psyllic acid, geddic acid,ceroplastic acid, hexatriacontylic acid, heptatriacontanoic acid,octatriacontanoic acid, α-linolenic acid, stearidonic acid,eicosapentaenoic acid, docosahexaenoic acid, linoleic acid, γ-linolenicacid, dihomo-γ-linolenic acid, arachidonic acid, docosatetraenoic acid,palmitoleic acid, vaccenic acid, paullinic acid, oleic acid, elaidicacid, gondoic acid, erucic acid, nervonic acid, or mead acid.

In a particular embodiment of the lipid supplement, the one or morelipids is not arachidonic acid or α-linolenic acid, or both.

In a different embodiment of the lipid supplement, the lipid supplementcomprises only one lipid. The only one lipid may be selected from thegroup comprising: a fatty acid; a glycerolipid; a glycerophospholipid; asphingolipid; a sterol lipid; a prenol lipid; a saccharolipid; or apolyketide.

In other embodiments of the lipid supplement, the only one lipid mayinclude a lipid-like substance, such as a poloxamer. The poloxamer maybe Kolliphor™, Synperonics™, or Pluronics™. In certain embodiments, thelipid-like substance may be Kolliphor™ P188.

In a specific embodiment of the lipid supplement, the only one lipid maybe a fatty acid. In such embodiment, the fatty acid may be a saturatedfatty acid. The saturated fatty acid may be selected from the groupcomprising propionic acid, butyric acid, valeric acid, caproic acid,enanthic acid, caprylic acid, pelargonic acid, capric acid, undecylicacid, lauric acid, tridecylic acid, myristic acid, pentadecylic acid,palmitic acid, margaric acid, stearic acid, nonadecylic acid, arachidicacid, heneicosylic acid, behenic acid, tricosylic acid, lignoceric acid,pentacosylic acid, cerotic acid, heptacosylic acid, montanic acid,nonacosylic acid, melissic acid, henatriacontylic acid, lacceroic acid,psyllic acid, geddic acid, ceroplastic acid, hexatriacontylic acid,heptatriacontanoic acid, or octatriacontanoic acid.

In a different such embodiment, the fatty acid may be an unsaturatedfatty acid. The unsaturated fatty acid may be selected from the groupcomprising α-linolenic acid, stearidonic acid, eicosapentaenoic acid,docosahexaenoic acid, linoleic acid, γ-linolenic acid,dihomo-γ-linolenic acid, arachidonic acid, docosatetraenoic acid,palmitoleic acid, vaccenic acid, paullinic acid, oleic acid, elaidicacid, gondoic acid, erucic acid, nervonic acid, or mead acid.

In a specific embodiment of the lipid supplement, the only one lipid maybe selected from the group comprising Mead's acid, arachidic acid,palmitoleic acid, oleic acid, myristic acid, palmitic acid, myristoleicacid, linoleic acid, stearic acid, α-linolenic acid, arachidonic acid,cholesterol, DL-α-tocopheryl, Kolliphor P188.

In a more specific embodiment of the lipid supplement, the only onelipid may be selected from the group comprising palmitic acid, stearicacid, oleic acid, linoleic acid, and α-linolenic acid.

In a still more specific embodiment of the lipid supplement, the onlyone lipid is either palmitic acid or oleic acid.

In a particular embodiment of the lipid supplement, the only one lipidis not arachidonic acid or α-linolenic acid.

Certain lipid profiles may be better suited to the culture of certaincell types. In one embodiment, a lipid profile higher in oleic acid,palmitic acid, and linoleic acid than in stearic acid and α-linolenicacid may enhance the survival or proliferation of one or more mammalianstem cells. In the same or a different embodiment, a lipid profile forenhancing the survival or proliferation of one or more mammalian stemcells comprises one or more of oleic acid, palmitic acid, linoleic acid,stearic acid, and/or α-linolenic acid at a level higher than any otherof the one or more lipids.

The skilled person will appreciate that using routine trial and error itwould be possible to identify beneficial or detrimental one or morelipids, whether singly or in combination, for use in a lipid supplementto enhance the survival or proliferation of one or more cells.

It may be further desired to load each of the one or more lipids onto acarrier at the same or different concentrations. For example, the one ormore lipids may be loaded onto the carrier to a concentration rangingfrom 1 ng/mL to 35 ug/mL. In the alternative, the one or more lipids maybe loaded onto the carrier in accordance with the narrowed concentrationrange as indicated in Table 1.

In order to enhance the proliferation or survival of one or moremammalian stem cells, a carrier may be required to transport some or allof the lipid supplement to such cells, whether in vitro or in vivo.Thus, a lipid supplement for enhancing the survival or proliferation ofone or more mammalian stem cells may comprise one or more lipids in thepresence of a carrier.

Such a carrier could be any biological or non-biological agent,substance, composition or complex that is capable of transporting someor all of the lipid supplement to a cell. More specifically, the carrieris capable of transporting one or more lipids of the lipid supplement toa cell.

By way of non-limiting examples, a carrier may be an albumin, a micelle,a liposome, an extracellular vesicle, an exosome, a cyclodextrin, ananostructured lipid carrier, or otherwise.

In embodiments where the carrier is an albumin, the albumin may be fromany source. Many types of albumin are known in the field of cellculture. In addition, particular albumins may be better suited forculturing stem cells. The different types of albumins may vary dependingon factors, such as their origin. For example, the albumin may be abovine albumin (BSA), a human albumin (HSA), or otherwise. Or, thealbumin may be a recombinant albumin. For example, the recombinantalbumin may be a recombinant human albumin (rHA) or a recombinant bovinealbumin (rBA).

In embodiments where the carrier is a liposome or an extracellularvesicle, such as an exosome, the one or more lipids may be presentwithin the lipid bilayer thereof, or as cargo packaged within aninternal space bounded by the lipid bilayer thereof.

Carriers of the disclosure may be purchased from a commercial entity ormay be isolated/synthesized using commercially available products orreagents. For carriers that are naturally-occurring, such asextracellular vesicles (including exosomes), liposomes, micelles, andalbumins, for example, such naturally-occurring carriers may be isolatedusing any established/available protocol. For carriers that are notnecessarily naturally-occurring but are readily synthesizable, such asliposomes, micelles, cyclodextrins, nanostructured lipid carriers, orotherwise, such not naturally-occurring carriers may be synthesizedusing any established/available protocol.

Some or all carriers may be purchased from a commercial entity.Specifically, powdered or solubilized albumin may be purchased from anyvendor, such as ThermoFisher Scientific, Sigma-Aldrich, or otherwise.

Whether isolated, manufactured or purchased, the carriers of thedisclosure may from the outset comprise one or more lipids or fattyacids. Such one or more lipids or fatty acids may be present atappreciable levels or at levels either very close to a detection limitor below the detection limit, and therefore at essentially undetectablelevels.

Where a specific lipid profile is required to affect a desired outcome,the presence of one or more lipids or fatty acids in association withthe carrier at the outset may be problematic if, for example, the lipidprofile is not amenable to affecting the desired outcome. Accordingly,in certain embodiments it may be desirable to remove or deplete some orall of such one or more lipids or fatty acids from the carrier.

In another embodiment, the one or more lipids may be loaded onto alipid-free or a lipid-reduced carrier, such as an albumin. Regardless ofwhether or not the carrier is lipid-free, lipid-reduced, or otherwise,the carrier may be loaded with a desired one or more types of lipids,generating a lipid-enriched carrier having a defined lipid signature.

In other embodiments, whether isolated, synthesized, or purchased, thecarrier may be free of lipids or fatty acids or substantially free oflipids or fatty acids.

In one embodiment of enhancing the survival or proliferation of one ormore mammalian stem cells, the one or more lipids (or the at least onefatty acid) and/or lipid-like substances may be pre-loaded onto acarrier. The one or more lipids (or the at least one fatty acid) and/orlipid-substances may be pre-loaded onto the carrier using anyconventional technique known in the field. For example, pre-loading theone or more lipids and/or lipid-substances onto the carrier may becarried out by combining a desired amount of each desired one or morelipids (or at least one fatty acid) and/or lipid-substances with adesired amount of the carrier and incubating such combination for a timesufficient to allow the one or more lipids (or at least one fatty acid)and/or lipid-substances and the carrier to come to an equilibrium, orsubstantially to an equilibrium. Exposing one or more mammalian stemcells to a thusly prepared carrier pre-loaded with one or more lipids(or at least one fatty acid) and/or lipid-substances may enhance thesurvival or proliferation of the one or more mammalian stem cells.

In another embodiment, the one or more lipids (or the at least one fattyacid) and/or lipid-substances may be combined but not necessarilypre-loaded onto the carrier. For example, combining the one or morelipids (or the at least one fatty acid) and/or lipid-substances with thecarrier may be carried out by combining a desired amount of each desiredone or more lipids (or at least one fatty acid) and/or lipid-substanceswith a desired amount of the carrier without allowing the carrier andthe one or more lipids (or at least one fatty acid) and/orlipid-substances to come to an equilibrium. Such a combination of theone or more lipids (or at least one fatty acid) and the carrier may beincubated without allowing the combination to come to an equilibrium.Exposing one or more mammalian stem cells to a thusly combined carrierand one or more lipids (or at least one fatty acid) and/orlipid-substances may enhance the survival or proliferation of the one ormore mammalian stem cells.

In another embodiment, the one or more lipids (or the at least one fattyacid) and/or lipid-substances may be provided separate from the carrierto a cell culture of one or more mammalian stem cells. Providing the oneor more lipids (or the at least one fatty acid) and/or lipid-substancesseparate from carrier to a cell culture of one or more mammalian stemcells can be accomplished in any order. For example, the carrier mayalready be present in the cell culture and the one or more lipids (orthe at least one fatty acid) and/or lipid-substances are addedafterward. Or, the one or more lipids (or the at least one fatty acid)and/or lipid-substances may already be present in the cell culture andthe carrier is added afterward. Or, neither the carrier nor the one ormore lipids and/or lipid-substances are already present in the culture,and both components may be added in any sequence with any or no timedelay between addition steps to the cell culture.

In certain embodiments, free one or more lipids (or at least one fattyacid) and/or lipid-substances may be spiked into a cell culture alreadycomprising a carrier. Spiking the free one or more lipids and/orlipid-substances could be achieved using any quantity and/or combinationof the free one or more lipids, provided that the spiked free one ormore lipids and/or lipid-substances is not detrimental to the survivalor proliferation of the one or more mammalian stem cells in the cellculture. Preferably, the spiked free one or more lipids and/orlipid-substances enhance the survival or proliferation of the one ormore mammalian stem cells in the cell culture.

In a further embodiment, it may be advantageous to spike additionalcarrier into the cell culture along with the free one or more lipids (orat least one fatty acid) and/or lipid-substances.

Culture Media

In one aspect of this disclosure culture media for enhancing thesurvival or proliferation of one or more cells cultured therein isprovided. In certain embodiments the one or more cells may be one ormore mammalian cells, and more specifically the one or more cells may beone or more mammalian stem cells. Various embodiments of a culturemedium for enhancing the survival or proliferation of one or more cellsare further described below.

In one embodiment, the culture medium comprises a lipid supplement asdescribed herein. For example, the lipid supplement may comprise one ormore lipids and/or lipid-substances, one or more lipids including atleast one fatty acid, one or more lipids including more than one fattyacid, or only one lipid.

In the same embodiments or in a different embodiment of the culturemedium, the lipid supplement may comprise one or more lipids and/orlipid-substances, one or more lipids including at least one fatty acid,one or more lipids including more than one fatty acid, or only onelipid, in the presence of a carrier.

In a specific embodiment of the culture medium, the one or more lipidsand/or lipid-substances, the one or more lipids including at least onefatty acid, the one or more lipids including more than one fatty acid,or the only one lipid, in the presence of a carrier is a lipid-enrichedcarrier. The lipid-enriched carrier may be a lipid-enriched albumin.

In one embodiment of the lipid supplement of the culture medium, the oneor more lipids and/or lipid-substances, the one or more lipids includingat least one fatty acid, the one or more lipids including more than onefatty acid, or the only one lipid may be pre-loaded onto the carrier andadded to culture medium whether prior to or after exposing the cells tothe culture medium. The lipid-enriched carrier, such as a lipid-enrichedalbumin, may be prepared using known methods wherein powdered orsolubilized carrier may be loaded with one or more lipids in accordancewith the description below.

In another embodiment of the lipid supplement of the culture medium, theone or more lipids, the one or more lipids including at least one fattyacid, the one or more lipids including more than one fatty acid, or theonly one lipid may be combined with a carrier in the culture medium, orin a separate solution, prior to exposing the cells to the culturemedium. Or, the one or more lipids, the one or more lipids including atleast one fatty acid, the one or more lipids including more than onefatty acid, or the only one lipid, and a carrier may each be added tothe culture medium separately after the culture medium has been added tothe cells.

In one embodiment, the culture medium comprises a lipid supplement asdescribed herein and one or more survival factors. The one or moresurvival factors may be any molecule, compound, or otherwise thatenhance the survival of the cultured mammalian stem cell. In turn, amammalian stem cell having enhanced survival, may also demonstrateenhanced proliferation in the disclosed culture medium.

In one embodiment, the one or more survival factors may comprise one ormore small molecule inhibitors. In certain embodiments, the one or moresmall molecule inhibitors may comprise one or more of Thiazovivin,Y-27632, CHIR99021, SB202190, MI-7, Necrostatin-1, NS3694, Wnt-059,NSCI, or BIPV5. In other embodiments, the one or more small moleculeinhibitors may comprise a Rho/Rock pathway inhibitor. One example of acommon Rho/Rock inhibitor is Y-27632.

The one or more survival factors of a disclosed culture medium, may bepresent in a concentration ranging from 1 nM to 1 mM. In a particularembodiment, the one or more survival factors such as Y-27632 is presentat a concentration of 10 μM.

In another embodiment, the culture medium for enhancing the survival orproliferation of one or more mammalian stem cells may further comprisean extracellular matrix. The extracellular matrix may be a naturallyderived matrix product, such as by way of non-limiting example, aproduct secreted by a cell or tissue. Or, the extracellular matrix maybe a decellularized matrix.

In some embodiments, the extracellular matrix may comprise one or moremonomatrix components. Non-limiting examples of monomatrix componentsinclude fibronectin, collagen, laminin, elastin, vitronectin, entactin,heparin sulphate, or proteoglycans alone or in combination. In otherembodiments the extracellular matrix may be Matrigel™.

In embodiments where the culture medium comprises an extracellularmatrix, a concentration of the extracellular matrix may be below agelation threshold thereof. The gelation threshold may vary depending onthe type of extracellular matrix (or matrix of one or more monomatrixcomponents) added to the culture medium. Notwithstanding, the gelationthreshold of an extracellular matrix (whether consisting of a monomatrixcomponent or comprising one or more monomatrix components) is the pointat which the culture medium forms a solid or substantially solidsolution, rather than a liquid or a semi-solid solution. In embodimentswhere the extracellular matrix is Matrigel™, the gelation threshold isabout 0.5% v/v or higher.

In another aspect, the present disclosure provides a culture medium forenhancing the survival or proliferation of one or more mammalian stemcells comprising an extracellular matrix component or components, andoptionally comprising a lipid supplement as described herein.

According to one embodiment of such culture medium, the extracellularmatrix may be a naturally derived matrix product, such as by way ofnon-limiting example, a product secreted by a cell or tissue. Or, theextracellular matrix may be a decellularized matrix.

In some embodiments, the extracellular matrix may comprise one or moremonomatrix components. Non-limiting examples of monomatrix componentsinclude fibronectin, collagen, laminin, elastin, vitronectin, entactin,heparin sulphate, or proteoglycans alone or in combination. In otherembodiments the extracellular matrix may be Matrigel™.

In embodiments where the culture medium comprises an extracellularmatrix, a concentration of the extracellular matrix may be below agelation threshold thereof. The gelation threshold may vary depending onthe type of extracellular matrix (or matrix of one or more monomatrixcomponents) added to the culture medium. Notwithstanding, the gelationthreshold of an extracellular matrix (whether consisting of a monomatrixcomponent or comprising one or more monomatrix components) is the pointat which the culture medium forms a solid or substantially solidsolution, rather than a liquid or a semi-solid solution. In embodimentswhere the extracellular matrix is Matrigel™, the gelation threshold isabout 0.5% v/v or higher.

In one embodiment, the culture medium may further comprise one or moresurvival factors. The one or more survival factors may be any molecule,compound, or otherwise that enhance the survival of the culturedmammalian stem cell(s). In turn, a mammalian stem cell having enhancedsurvival, may also demonstrate enhanced proliferation in the disclosedculture medium.

Further details of the one or more survival factors may be gleaned fromthe description thereof hereinabove.

The culture media will also contain other factors necessary for thegrowth and survival of the stem cells. Media formulations or base mediaformulations appropriate for culturing particular types of mammalianstem cells are commercially available. Any such media formulation orbase media formulation may be used to formulate the medium disclosedherein, and to carry out the methods disclosed herein.

In one embodiment, the culture medium may comprise growth factors thatsupport the culture of mammalian cells.

In a particular embodiment applicable to hPSC, the growth factors mayinclude, but are not limited to, SCF, EGF, TGFβ, FGF, LIF, and BMP.

The culture medium may also comprise other additives that support theculture of mammalian stem cells. In another embodiment, applicable tohuman stem cells, the other additives may include, but are not limitedto, 4-aminobutyric acid, BSA, pipecolic acid, and lithium chloride.

Methods

Stem cells are commonly cultured in vitro. In such in vitroapplications, it is preferable to culture stem cells under particularculture conditions. If in vitro stem cells are not cultured underparticular culture conditions, the stem cells may grow sub-optimally. Insome cases sub-optimal growth may comprise a decreased growth rate. Inother cases, sub-optimal growth may comprise unintended differentiationof the stem cells. In still other cases, sub-optimal growth may comprisecell death, such as by apoptosis, necrosis, autophagy, or otherwise. Theinventors have shown that culturing stem cells in a culture mediumcomprising a lipid supplement may enhance the survival or proliferationof one or more mammalian stem cells.

In one aspect, the present disclosure provides methods of enhancing thesurvival or proliferation of mammalian stem cells comprising culturingthe stem cells in a culture medium comprising a lipid supplement asdescribed herein.

In one embodiment, the present disclosure provides methods of enhancingthe survival or proliferation of mammalian stem cells comprisingculturing the stem cells in a culture medium comprising a lipid enrichedcarrier as described herein.

In a more specific embodiment, the present disclosure provides methodsof enhancing the survival or proliferation of mammalian stem cellscomprising culturing the stem cells in a culture medium comprising alipid-enriched albumin.

In another embodiment, the culture media may also comprise other factorssuch as one or more survival factors as described herein.

In another embodiment, the present disclosure provides a method ofenhancing the survival or proliferation of one or more mammalian stemcells comprising culturing the stem cells in a culture medium furthercomprising an extracellular matrix component or components.

In another aspect, the present disclosure provides a method of enhancingthe survival or proliferation of mammalian stem cells comprisingculturing the stem cells in a culture medium comprising an extracellularmatrix component or components and optionally a lipid supplement asdescribed herein.

In specific embodiments, the extracellular matrix component orcomponents are provided below a gelation threshold thereof.

In one embodiment, the culture media may also comprise other factorssuch as one or more survival factors as described herein.

Accordingly, the present disclosure provides for methods of enhancingthe survival or proliferation of one or more mammalian stem cells in aculture medium according to a medium as described hereinabove. Enhancedsurvival or proliferation may be calculated by any technique known inthe art, including but not limited to determining a % cloning efficiencyby dividing the number of colonies/clones generated by the number ofinput cells or clumps/clusters thereof. In certain embodiments, enhancedsurvival or proliferation comprises yielding a 5%-65% cloningefficiency. Or, enhanced survival or proliferation may be referenced byindicating the number of recovered clones/colonies from the inputtedcells or clumps/clusters thereof.

The stem cells can be any mammalian stem cell that one wishes to cultureto enhance the survival and proliferation of the cells.

In this disclosure, stem cells can be any mammalian stem cell. In oneembodiment, the stem cells are non-rodent. For example, the non-rodentmammalian stem cells may be porcine stem cells. In the alternative, thenon-rodent mammalian stem cells may be primate stem cells. In thefurther alternative, the primate stem cells may be human stem cells.

The skilled person will also be aware that the mammalian stem cells maycorrespond to any developmental stage of the mammal. For example, themammalian stem cells may be embryonic in origin, such as embryonic stemcells (ESC). In the alternative, the mammalian stem cells may originatefrom a tissue or organ of an adult mammal. Or, the mammalian stem cellmay be an induced pluripotent stem cell (iPSC), wherein the iPSC may begenerated using any technique known in the art. Collectively, ESC andiPSC are termed pluripotent stem cells (PSC).

In view of the potential downstream applications of in vitro culturedmammalian stem cells, particularly those cultured by seeding one or moremammalian stem cells as single cells, it may be desirable that themammalian stem cells have a normal karyotype. It may be furtherdesirable that the normal karyotype is stable. A normal karyotype may becharacterized by an appropriate number of chromosomes characteristic fora species. In addition or in the alternative, a normal karyotype may becharacterized by a proper staining profile, using any stain known in theart for banding analysis of chromosomes, such as Giemsa. In furtheraddition or in the further alternative, a normal karyotype may becharacterized by properly sized chromosomes.

In another embodiment, the mammalian stem cells are geneticallyengineered. Mammalian stem cells may be genetically engineered using anytechnology known in the art. For example, a mammalian stem cell may begenetically engineered using gene editing technology. Gene editingtechnology may include, but is not limited to, CRISPR technology,zinc-finger nuclease technology, TALEN technology or ARCUS technology.

The problems associated with the expansion and/or survival of mammalianstem cells are heightened when it is desirable to genetically-engineerthe mammalian stem cells. For example, when one or more mammalian stemcells are subjected to gene editing technology, it is likely that no twomammalian stem cells are identically genetically-engineered. However, ifsuch genetically-engineered mammalian stem cells will be used indownstream applications, regardless of whether the applications are invivo or in vitro, it is preferable to use a clonal population thereofobtained, for example, by seeding one or more mammalian stem cells assingle cells.

The disclosed methods may comprise providing a population or culture ofmammalian stem cells. Or, the disclosed methods may comprise providingone or more mammalian stem cells. The provided mammalian stem cells mayhave been maintained in any culture media known in the art. The type ofculture media for maintaining the mammalian stem cells will depend onthe nature of the mammalian stem cells. As indicated above, themammalian stem cells may be embryonic in origin. Alternatively, themammalian stem cells may originate from an adult tissue or organ. In thefurther alternative, the mammalian stem cell may have been induced ortransdifferentiated from a suitable parental cell. The type of mediumused to maintain the mammalian stem cell will also depend on the speciesfrom which the mammalian stem cell originates. For example, human PSC(hPSC), may be maintained in an mTeSR™ media formulation.

In one embodiment, the stem cells are cultured for 6-8 days afterpassaging and then are dissociated to single cells and seeded into theculture medium comprising the lipid supplement and optionally the one ormore survival factors. In a specific embodiment, the cells are seeded ata density of 1 cell/well up to 1000 cells/well. The cells are then fedagain at day 2 with the medium comprising the lipid supplement andoptionally the one or more survival factors then fed at day 4 with theregular growth medium (total of 4 days in cloning supplement (ie. lipidsupplement)). The cells can then be fed daily until the colonies areharvested, such as around 7-12 days.

In another embodiment, the stem cells may be cultured for 6-8 days afterpassaging and then dissociated to single cells and seeded into theculture medium comprising one or both of a lipid supplement or anextracellular matrix component or components, and optionally the one ormore survival factors. In a specific embodiment, the cells may be seededat a density of 1 cell/well up to 1000 cells/well. The cells may then befed again at day 2 with the medium comprising one or both of the lipidsupplement or the extracellular matrix component or components, andoptionally the one or more survival factors then fed at day 4 with theregular growth medium (total of 4 days in cloning supplement (ie. lipidsupplement)). The cells can then be fed daily until the colonies areharvested, such as around 6-12 days.

The skilled person will be aware that the number of days for exposingthe one or more mammalian stem cells to the lipid supplement and/or theextracellular matrix component or components is merely a guideline andcould readily be varied. Such exposure may be longer or shorter induration than the 2 days suggested above. For example, such exposurecould be less than 2 days, such as approximately 36 hours, 24 hours, 18hours, 12 hours, 8 hours, 6 hours, 4 hours, 2 hours, 1 hour, or less.Or, such exposure could be for longer than 2 days. For example, thelonger than 2 day exposure may be up to the total amount of time the oneor more mammalian stem cells may be cultured in the same medium, withoutundergoing a change of medium.

Further, exposure of the one or more mammalian stem cells to the lipidsupplement and/or the extracellular matrix component or components mayoccur any number of times. For example, the one or more mammalian stemcells may only require a single exposure to the lipid supplement and/orextracellular matrix component or components. Such single exposure couldbe for any appropriate amount of time, as specified above. Or, the oneor more mammalian stem cells may require two or more exposures to thelipid supplement and/or extracellular matrix component or components.

In another specific embodiment, the cells may be fed daily via fedbatchfeeding at day 2 with the regular growth medium or a medium comprisingone or both of the lipid supplement or the extracellular matrixcomponent or components, and optionally the one or more survivalfactors. The cells can then be fed daily until the colonies areharvested, such as around 4-10 days.

The provided mammalian stem cells may be sub-cultured once the mammalianstem cells achieve threshold confluency. Or, the provided mammalian stemcells may be sub-cultured as dictated by colony health, such as may bedetermined by colony appearance, size or morphology.

Sub-culturing the provided mammalian stem cells may be performed usingany technique, appropriate to the particular culture of mammalian stemcells, known in the art of stem cell culture. For example, once hPSCachieve a confluency of approximately 70% it may be desirable tosub-culture the cells into a different culture vessel.

Typically, an hPSC culture at an appropriate level of confluency and/orhaving a particular colony size may be detached from a culture vessel byapplying a suitable agent. The agent may comprise digestive enzyme(s) orchemicals known in the field to detach colonies. If a digestiveenzyme(s) is used, it may be desirable to inactivate the digestiveenzyme(s) by the addition of a second inactivating solution. In order tofurther disaggregate the detached mammalian stem cells, it may benecessary to expose them to an agitative force, such as by repetitiveupward and downward pipetting or by a technician mechanically strikingthe culture vessel.

Upon sufficient agitation, the detached, disaggregated mammalian stemcells may exist as a single cell suspension or as clusters havingdesirable dimensions, which may comprise a desired cell number range.Sufficiently disaggregated cells may be sub-cultured at a desired celldensity, by plating one or more of the mammalian stem cells in anappropriate culture vessel.

Plating the desired cell density of the one or more mammalian stem cellsmay be performed using any known method. A single cell suspension of themammalian cells or a suspension of cell clusters may be plated usingconventional techniques. For example, after determining the cell densityof the suspension, an appropriate volume of the suspension may be usedto seed a culture vessel, or a well or microwell thereof. Or, the cellsuspension can be subjected to fluorescence activated cell sorting, andthe sorted mammalian cells may be appropriately partitioned, at anappropriate cell number, into a culture vessel, or a well or microwellthereof.

In certain applications, it may be desirable to seed the mammalian stemcells by plating only a single mammalian cell into a culture vessel, ora well or microwell thereof. Or, it may be desirable to seed themammalian stem cells by plating single mammalian cells at a sufficientlylow density into a culture vessel, or a well or microwell thereof. Insuch circumstances, it is desirable that the cell density issufficiently low to minimize the tendencies of the plated mammaliancells to aggregate, such as by ensuring sufficient spacing between cellsin the culture vessel, or a well or microwell thereof. Also, suchsufficient spacing may minimize paracrine signaling among the mammalianstem cells in the culture vessel, or well or microwell thereof.

In one embodiment, a single hPSC may be plated in a single well ormicrowell of a culture vessel. The single hPSC may be plated afterhaving determined the cell density of the detached, disaggregatedpopulation of hPSC and plating an appropriate volume. Or, the singlehPSC may be plated using cell sorting technology, such as fluorescenceactivated cell sorting. In another embodiment, one or more hPSC may beplated at a clonal density in a relatively larger culture vessel, orwell thereof. For example, a clonal density may comprise a density ofbetween 1 cell/well to 1000 cells/cm².

Overall, the presently disclosed methods may encompass culturing the oneor more mammalian stem cells in a culture medium of this disclosure as amonolayer (ie. adherent culture) or as a non-adherent-culture (ie. insuspension). Regardless of whether the one or more mammalian stem cellsare seeded as a monolayer or as a non-adherent culture, the disclosedmethods may further comprise seeding the one or more cells at a seedingdensity of 1 cell/well up to about 1000 cells/cm²). In some embodimentsthe one or more mammalian stem cells may be seeded as a single cell.

In embodiments wherein the one or more mammalian stem cells are culturedas a monolayer, culturing as a monolayer may comprise seeding the one ormore cells in an extracellular matrix.

In another embodiment, mammalian stem cells may be plated, whether assingle cells or as clusters, with a view to culturing the one or moreplated mammalian cells either as a suspension or an adherent culture.

Where it may be desired to culture the mammalian stem cells as asuspension culture, whether as single cells or as clusters, it may bedesirable to seed mammalian cells as clumps of cells or a single cellsuspension directly into a bioreactor, spinner flask, suspension cultureplate or other such vessel that promotes the growth of stem cells insuspension. It may also be desirable to pre-aggregate the stem cellsusing micro-well plates or any other such method to create equally sizedaggregates before plating stem cells into the aforementioned vessels. Itmay also be desirable to culture the cells in the presence of microcarriers to support the growth of stem cells in suspension.

Where it may be desired to culture the mammalian stem cells as anadherent culture, whether as single cells or as clusters, it may bedesirable to plate the one or more mammalian stem cells onto a suitablematrix. The matrix may be any matrix that supports the culture of theone or more mammalian stem cells. The matrix may also promote attachmentof the one or more mammalian stem cells. For example, the matrix maycomprise extracellular matrix proteins that support the culture of theone or more mammalian stem cells. Various matrices comprisingextracellular matrix proteins are commercially available, such asMatrigel. Examples of some extracellular matrix proteins contemplated inthis disclosure include laminin, collagen, fibronectin, vitronectin, orentactin. The matrix contemplated in this disclosure may furthercomprise known quantities of a combination of extracellular matrixproteins, such as laminin, collagen, fibronectin, vitronectin, entactin,or otherwise.

The plated one or more mammalian stem cells, whether cultured insuspension or as an adherent culture, should be supplemented with aculture medium that supports the culture thereof, such as a culturemedium of this disclosure. As indicated above, culture conditions mayvary depending on the nature and characteristics of the mammalian stemcells. The skilled person will understand that the culture medium forsupporting the culture of the mammalian stem cells should comprise abase medium formulation appropriate to the nature and characteristics ofthe mammalian stem cells.

In one embodiment, hPSC may be maintained and sub-cultured in a mTeSR™formulation, such as mTeSR™1, mTeSR™2, TeSR™-E8 or mTeSR™3D. mTeSR™formulations are well-suited to standard culturing of hPSC. In otherembodiments, hPSC may be maintained and sub-cultured in knock-out serumreplacement (KOSR) based medium, StemMACS™ iPS-Brew (Miltenyi Biotec)iPS-Brew, Essential 8™ Medium (Thermo Fisher Scientific), Essential-8,StemFlex™ Medium (Thermo Fisher Scientific), Cellartis® DEF-CS™ (Takara)or other medium to support the growth of stem cells.

In another embodiment, adult human stem cells, such as mesenchymal stemcells may be maintained and sub-cultured in MesenCult™-XF,MesenCult™-ACF, or MesenCult™-PL, for example.

In another embodiment, adult human stem cells, such as neural stem cellsmay be maintained and sub-cultured in NeuroCult™, BrainPhys™, or XcellNeural Medium, for example.

By culturing the one or more non-rodent mammalian cells with a mediumcomprising a lipid-enriched albumin and optionally one or more survivalfactors, it may be possible to yield a 5% to 65% survival rate for theone or more cells cultured with the medium.

TABLE 1 Concentration of fatty acids on lipid-enriched albumin. FattyAcid Narrowed Range Broad Range Mead's acid 0.006-0.371 μg/mL 1 ng/mL-35μg/mL Arachidic 0.054-1.457 μg/mL 1 ng/mL-35 μg/mL Palmitoleic0.054-1.817 μg/mL 1 ng/mL-35 μg/mL Oleic 0.965-33.576 μg/mL 1 ng/mL-35μg/mL Myristic 0.033-1.16.5 μg/mL 1 ng/mL-35 μg/mL Palmitic 1.115-35.129μg/mL 1 ng/mL-35 μg/mL Myristoleic 0.103-0.536 μg/mL 1 ng/mL-35 μg/mLLinoleic 1.734-23.907 μg/mL 1 ng/mL-35 μg/mL Stearic 0.273-3.979 μg/mL 1ng/mL-35 μg/mL alpha-linolenic 0.022-1.709 μg/mL 1 ng/mL-35 μg/mL

The following non-limiting examples are illustrative of the presentdisclosure:

EXAMPLES Example 1

12-well culture plates were coated with 0.5 mL of cloning matrix orextracellular matrix (1:25 dilution in CellAdhere™ Dilution Buffer) andplaced at room temperature for 1 hour. The matrix was then aspirated and1 mL of media (mTeSR™1 or TeSR™-E8™ supplemented with either 10 μMY27632 or media comprising lipid-enriched albumin, GABA, pipecolic acid,lithium chloride, FGF, TGFβ and Y-27632 (hereafter termed cloningsupplement)) was added to each well and the plate was placed at 37° C.for 1 hour. hPSC lines were dissociated to single cells and seeded intothe pre-warmed plates at 25 cells/cm² and placed at 37° C. for two days.The cells were then fed with fresh media (mTeSR™1 or TeSR™-E8™supplemented with either 10 μM Y-27632 or cloning supplement) and placedat 37° C. for two days. On day four the cells were then fed with 1 mL ofmTeSR™1 or TeSR™-E8™ without additives and fed daily until day seven.Cells were then fixed using 4% paraformaldehyde and stained for alkalinephosphatase. Undifferentiated colonies were then counted and cloningefficiency was determined using the following calculation: (Number ofundifferentiated colonies at day seven (per well)/Number of cells seededat day zero (per well))×100. (Error bars represent standard deviationfrom three biological replicates).

The results of this Example are shown in FIG. 1 and demonstrate thateach hPSC line (H1, H7, WLS-1C, STiPS-M001) plated at clonal density (25cells/cm²) shows enhanced cloning efficiency when grown in mTeSR™1 orTeSR™-E8™ when mTeSR™1 and TeSR™-E8™ are supplemented with cloningsupplement, but not when grown in mTeSR™1 or TeSR™-E8™ supplemented withRock inhibitor alone.

Example 2

96-well culture plates were coated with 50 μL of cloning matrix orextracellular matrix (1:25 dilution in CellAdhere™ Dilution Buffer) andplaced at room temperature for 1 hour. The matrix was then aspirated and100 μL of media (mTeSR™1 supplemented with either 10 μM Y-27632 orcloning supplement was added to each well and the plate was placed at37° C. for 1 hour. hPSC lines were dissociated to single cells andsorted using a BD FACSAria™ Fusion at 1 cell/well and placed at 37° C.for two days. The cells were then fed with fresh media (mTeSR™1supplemented with either 10 μM Y-27632 or cloning supplement) and placedat 37° C. for two days. On day four the cells were then fed with 100 μLmTeSR™1 without additives and fed daily until day seven. Cells were thenfixed using 4% paraformaldehyde and stained for alkaline phosphatase.Undifferentiated colonies were then counted and cloning efficiency wasdetermined using the following calculation: (Number of undifferentiatedcolonies (per plate)/Number of wells seeded (at 1 cell per well))×100.(Error bars represent SEM from two technical replicates).

The results of this Example are shown in FIG. 2 and demonstrate thateach hPSC line (H1, H7, WLS-1C, STiPS-M001) plated at a density of onecell per well shows enhanced cloning efficiency when grown in mTeSR™1 orTeSR™-E8™ when mTeSR™1 and TeSR™-E8™ are supplemented with cloningsupplement, but not when grown in mTeSR™1 or TeSR™-E8™ supplemented withRock inhibitor alone.

Example 3

Eight independent WLS-1C clones were manually picked 10 days followingsingle cell deposition (from Example 2) and expanded for 5 passagesusing mTeSR™1 and passaged using Gentle Cell Dissociation Reagent (fullprotocol available stemcell.com). Total number of clumps per well werecounted at the end of each passage to determine the daily fold expansionof the cloned lines compared to the clump passaged control. Daily foldexpansion was determined using the following calculation: (total numberof clumps at the end of passage/number of colonies seeded at thebeginning of passage)/number of days in culture. (Error bars representdata from at least two biological replicates).

The results of this example are shown in FIG. 3 and demonstrate thateight independent WLS-1C clones formed under conditions described inExample 2 exhibit a daily fold expansion comparable to hPSC culturedconventionally as clumps.

Example 4

12-well culture plates were coated with 0.5 mL of cloning matrix orextracellular matrix (1:25 dilution in CellAdhere™ Dilution Buffer) andplaced at room temperature for 1 hour. The matrix was then aspirated and1 mL of test media was added to each well. The different test mediaconsisted of control media (mTeSR™1 supplemented with 10 μM Y-27632), ormTeSR™1 supplemented with media comprising cloning supplement made withlipid enhanced BSA, or mTeSR™1 supplemented with cloning supplement withlipid stripped BSA that had either been lipid loaded with ethanol only,three fatty acids or five fatty acids. The plate was then placed at 37°C. for 1 hour. hPSC lines were dissociated to single cells and seededinto the pre-warmed plates at 25 cells/cm² and placed at 37° C. for twodays. The cells were then fed with the corresponding test media andplaced at 37° C. for two days. On day four the cells were then fed with1 mL of mTeSR™1 without additives and fed daily until day eight. Cellswere then fixed using 4% paraformaldehyde and stained for alkalinephosphatase. Undifferentiated colonies were then counted and cloningefficiency was determined using the following calculation: (Number ofundifferentiated colonies at day seven (per well)/Number of cells seededat day zero (per well))×100. (Error bars represent SEM from threetechnical replicates).

The results of this example are shown in FIG. 4 and demonstrate that H1hPSC grown in mTeSR™1 supplemented with Rock inhibitor and alipid-stripped BSA loaded with either oleic, palmitic, and linoleic(3FA) or oleic, palmitic, linoleic, stearic and alpha-linoleic (5FA) at600 μg/g (per fatty acid), exhibit enhanced cloning efficiencycomparable to a purchased lipid enhanced BSA, but not for lipid strippedBSA loaded with ethanol (control).

Example 5

Representative images of wells from Example 4 (Lipid-stripped BSA 1)stained with alkaline phosphatase (Far Red) and imaged using ImageXpressMicro.

The results of this example show that the colonies that were generatedin Example 4 display comparable colony size/morphology to coloniesgenerated in commercially available lipid enhanced BSA.

Example 6

The results of this example are shown in FIG. 6 and demonstrate thatSTiPS-M001, H7, and H9 hPSCs cultured under non-adherent conditions inmedium comprising Y-27632 and either lipid enriched albumin or 0.2%Matrigel® exhibit a higher number of recovered clones per well than whencultured with lipid enriched albumin-free media and Y-27632. Theseresults further show that medium comprising Y-27632, lipid enrichedalbumin and 0.2% Matrigel® exhibit a substantial and synergistic effect,with a much higher number of recovered clones per well than would beexpected by adding the recoveries observed using medium containing anysingle of these components.

Example 7

The results of this example are shown in FIG. 7 and demonstrate thatSTiPS-M001, WLS-1C, H7, and H9 hPSCs cultured under non-adherentconditions in medium comprising Y-27632 and either lipid enrichedalbumin or lipid enriched albumin and 0.2% Matrigel® exhibit a highercloning efficiency (number of clones recovered divided by number ofcells seeded) per well than when cultured with lipid enrichedalbumin-free media and Y-27632.

Example 8

The results of this example are shown in FIG. 8 and demonstrate thatWLS-1C, H1 and STiPS-F016 cells cultured in the presence of an otherwiselipid poor albumin (ie. substantially free of lipid) that has beenloaded with a single fatty acid, two fatty acids or three fatty acidsexhibit varying levels of cloning efficiency depending on the nature ofthe one or two fatty acids.

In particular, each of the one or more fatty acids was incubated withalbumin at a concentration of 600 μg of each fatty acid per gram ofalbumin. The product of such incubation was subsequently added to theculture medium at a final concentration of 6 ng/mL.

H1, WLS-1C, and STiPS-F016 hPSCs displayed comparable cloningefficiencies when treated with either oleic acid or palmitic acid, orboth, in comparison to the 3 fatty acid-loaded albumin (as shown in FIG.8 ).

Notably, loading only linoleic acid may reduce cloning efficiency inWLS-1C and H1 hPSCs below the levels of control and both oleic acidand/or palmitic acid.

Example 9

The results of this example are shown in FIG. 9 and demonstrate thatcloning efficiency may be enhanced by adding free fatty acid to a mediumcomprising additional low-lipid (ie. lipid-free or lipid-reduced)carrier.

WLS-1C, H1 and STiPS-F016 cells cultured in the presence of either threefree fatty acids (palmitic, oleic, and linoleic) plus carrier or analbumin loaded with three fatty acids (palmitic, oleic, and linoleic)exhibit comparable levels of cloning efficiency.

In particular, 600 μg of each fatty acid per gram of albumin wasincubated to yield the lipid-enriched albumin. Or, 600 μg of each freefatty acid per gram of additional low-lipid albumin were spiked into thecell culture. After 4 days of culture under these conditions (with amedium change at day 2) followed by 7 days of culture in standardmTeSR™1 the cloning efficiency of each cell line in each culturecondition was assessed.

It is notable that providing the three free fatty acids withoutadditional low-lipid carrier may also yield appreciable cloningefficiency levels.

Example 10

The results of this example are shown in FIG. 10 and demonstrate that,whether in mTeSR™1 (FIG. 10 a ) or in TeSR™E8™ (FIG. 10 b ), spikingfree fatty acids achieves better cloning efficiency for H1, 1C andSTiPS-F016 hPSCs when the free fatty acids are provided in the presenceof additional low-lipid carrier.

Specifically, when using a protein-reduced medium, such as TeSR™E8™, theinclusion of additional carrier is necessary to enhance the cloningefficiency of cells exposed to free fatty acids.

Example 11

The results of this example are shown in FIG. 11 and demonstrate thatthe cloning efficiencies of WLS-1C and H1 cells using two differentalbumins or albumin lots from 4 different suppliers are enhanced whenproviding said albumins along with two free fatty acids to the cultureof cells.

Notably, the cloning efficiencies using an albumin from one supplier (A1and A2) were not enhanced when providing said albumins along with twofree fatty acids. Of further note, albumins A1 and A2 yielded the lowestcloning efficiencies even without the presence of the additional twofree fatty acids. This observation may arise due to the high overalllevels of many fatty acids and/or the presence of one or more fattyacids detrimental to cloning efficiencies.

Example 12

The results of this example are shown in FIG. 12 and demonstrate theeffect of individual free fatty acids in the presence of a carrier onthe cloning efficiencies of H1 and WLS-1C hPSCs.

In this experiment, 600 μg of the shown free fatty acids were spikedinto culture medium (mTeSR™1 plus 10 μM Y-27632) per gram of carrieralso spiked into the culture medium. Cloning efficiencies werecalculated as described in Example 1, for example.

Whereas at least oleic acid, palmitic acid, myristic acid, stearic acidand cholesterol may be helpful in enhancing the survival andproliferation of H1 and WLS-1C hPSCs, it appears that the presence of atleast arachidonic acid and α-linolenic acids may be detrimental.

Example 13

The results of this example are shown in FIG. 13 and demonstrate thatloading a carrier with certain fatty acids is detrimental to cloningefficiencies of cells exposed thereto.

In the experiment shown in FIG. 13 , 300 μg/g of either arachidonic acidor α-linolenic acid were loaded onto a specific BSA sample and added toa culture comprising H1, WLS-1C, and H9 hPSCs. Each of the loaded fattyacids decreased the cloning efficiency of the tested BSA sample.

While the present invention has been described with reference to whatare presently considered to be the preferred examples, it is to beunderstood that the invention is not limited to the disclosed examples.To the contrary, the invention is intended to cover variousmodifications and equivalent arrangements included within the spirit andscope of the appended claims.

All publications, patents and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be

1. A culture medium for enhancing the survival or proliferation of oneor more mammalian stem cells, comprising one or more lipids and acarrier, wherein the one or more lipids is one or both of oleic acid andpalmitic acid, and wherein an amount of oleic acid and/or palmitic acidis greater than an amount of alpha-linolenic acid and/or arachidonicacid.
 2. The culture medium according to claim 1, wherein the one ormore lipids is selected from the group consisting of Mead's acid,arachidic acid, palmitoleic acid, myristic acid, myristoleic acid,linoleic acid, stearic acid, cholesterol, DL-alpha-tocopheryl andKolliphor P188.
 3. The culture medium according to claim 1, wherein thecarrier is an albumin, a liposome, an extracellular vesicle, an exosome,a nanostructured lipid carrier, or a cyclodextrin.
 4. The culture mediumaccording to claim 1, wherein a concentration of the one or more lipidsranges from 1 ng/mL to 35 μg/mL.
 5. The culture medium according toclaim 1, further comprising one or more survival factors.
 6. The culturemedium according to claim 5, wherein the one or more survival factorscomprise one or more small molecule inhibitors.
 7. The culture mediumaccording to claim 6, wherein the one or more small molecule inhibitorscomprise a Rho/Rock pathway inhibitor.
 8. The culture medium accordingto claim 7, wherein the Rho/Rock pathway inhibitor is Y-27632 at aconcentration ranging between 1 μM to 20 μM.
 9. The culture mediumaccording to claim 1, further comprising an extracellular matrix at aconcentration below a gelation threshold thereof.
 10. The culture mediumaccording to claim 9, wherein the gelation threshold is about 0.5% orhigher.
 11. The culture medium according to claim 10, wherein theextracellular matrix comprises one or more monomatrix components, andthe one or more matrix proteins comprise a: fibronectin, collagen,laminin, elastin, vitronectin, entactin, heparin sulphate, orproteoglycan.
 12. A method for enhancing the survival or proliferationof one or more mammalian stem cells, the method comprising culturing theone or more mammalian stem cells in a culture medium as claimed inclaim
 1. 13. The method according to claim 12, wherein culturingcomprises culturing as a monolayer.
 14. The method according to claim12, wherein culturing comprises culturing the one or more cells undernon-adherent conditions.
 15. The method according to claim 12, furthercomprising seeding the one or more cells at a seeding density of 1cell/well up to about 1000 cells/cm².
 16. The method according to claim12, further comprising yielding a survival rate of about 5% to about 65%for the one or more cells.
 17. The method according to claim 12, furthercomprising exposing the one or more mammalian stem cells to anextracellular matrix.
 18. The culture medium according to claim 17,wherein a concentration of the extracellular matrix in the medium isbelow a gelation threshold thereof.
 19. The method according to claim18, wherein the gelation threshold is about 0.5% or higher.
 20. Themethod according to claim 19, wherein the extracellular matrix comprisesone or more monomatrix components, and the one or more matrix proteinscomprise a: fibronectin, collagen, laminin, elastin, vitronectin,entactin, heparin sulphate, or proteoglycan.